Nad(p)- dependent responsive enzymes, electrodes and sensors, and methods for making and using the same

Abstract

NADP-dependent oxidoreductase compositions, and electrodes, sensors and systems that include the same. Analyte sensors include an electrode having a sensing layer disposed thereon, the sensing layer comprising a polymer and an enzyme composition distributed therein. The enzyme composition includes nicotinamide adenine dinucleotide phosphate (NAD(P)+) or derivative thereof, an NAD(P)+-dependent dehydrogenase; an NAD(P)H oxidoreductase; and an electron transfer agent comprising a transition metal complex.

What is claimed is: 
     
         1 . A sensor for detecting D-3-hydroxybutyrate in vivo, the sensor comprising:
 a first portion configured to be positioned above a user's skin; and   a second portion configured to be transcutaneously positioned in the user's skin and in contact with the user's interstitial fluid to monitor the level of D-3-hydroxybutyrate in vivo, the second portion comprising:   i) a working electrode having an enzyme composition disposed thereon, the enzyme composition comprising:   a) nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) or derivative thereof;   b) D-3-hydroxybutyrate dehydrogenase;   c) diaphorase;   d) an electron transfer agent;   e) a polymer,
 wherein the NAD(P)+ or derivative thereof is not covalently bonded to the polymer; and 
   ii) a mass transport limiting membrane disposed over the enzyme composition,   wherein the working electrode is configured to generate a signal associated with the level of D-3-hydroxybutyrate.   
     
     
         2 . The analyte sensor of  claim 1 , wherein the D-3-hydroxybutyrate dehydrogenase is covalently bonded to the polymer 
     
     
         3 . The analyte sensor of  claim 2 , wherein the diaphorase is covalently bonded to the polymer. 
     
     
         4 . The analyte sensor of  claim 3 , wherein the electron transfer agent is covalently bonded to the polymer. 
     
     
         5 . The analyte sensor of  claim 1 , wherein enzyme composition further comprises a crosslinker for immobilizing the enzyme composition onto a surface of the working electrode. 
     
     
         6 . The analyte sensor of  claim 1 , wherein the mass transport limiting membrane limits flux of D-3-hydroxybutyrate to the working electrode. 
     
     
         7 . The analyte sensor of  claim 6 , wherein the mass transport limiting membrane comprises crosslinked polymers. 
     
     
         8 . The analyte sensor of  claim 7 , wherein the polymers contain heterocyclic nitrogen groups. 
     
     
         9 . The analyte sensor of  claim 7 , wherein the polymers comprise polyvinylpyridine. 
     
     
         10 . The analyte sensor of  claim 7 , wherein the polymers comprise poly (4-vinylpyridine-co-styrene). 
     
     
         11 . The analyte sensor of  claim 8 , wherein the mass transport limiting membrane further comprises a hydrophilic or hydrophobic modifier. 
     
     
         12 . The analyte sensor of  claim 6 , wherein the mass transport limiting membrane is a polyurethane or polyether urethane membrane. 
     
     
         13 . The analyte sensor of  claim 6 , wherein the signal associated with the level of D-3-hydroxybutyrate increases linearly as a function of D-3-hydroxybutyrate concentration over a range of 0 mM to 10 mM. 
     
     
         14 . The analyte sensor of  claim 13 , wherein the signal and D-3-hydroxybutyrate concentration exhibit an R2 value of at least 0.99 over a range of 0 mM to 10 mM. 
     
     
         15 . The analyte sensor of  claim 6 , wherein the signal decays no more than 8% when exposed to a given hydroxybutyrate concentration for 45 hours.